Immunological rejection is the most common cause of corneal allo-transplant failure particularly when the recipient has a high risk vascularized (HRV) corneal bed. In fact, of the 40,000 patients per year receiving a CT for vision rehabilitation, 4-6,000 are considered HRV and the success rate is dismal (<30%). Induction of immune tolerance for these transplants is a critical and unmet medical need according to the NEI. (https://nei.nih.gov/news/scienceadvances/advances/corneal_transplantation). The studies proposed in this application will develop a unique combinatorial strategy never tested in a solid organ transplant to induce long- term tolerance in recipients of high-risk vascularized corneal transplants (HRVCT) through sequential: a) cyclophosphamide (Cy) deletion of antigen (Ag) specific effector T cells (Aim 1), b) immune suppression via marked in vivo Treg expansion (Aim 2) and c) inflammatory gene epigenetic regulation via bromodomain inhibitors BETi (Aim 3). Our preliminary data demonstrate that the use of brief, low dose cyclophosphamide (Cy) administered post-CT can dramatically prolong allograft survival by deleting effector T cells and diminishing corneal neovascularization. Our novel two-pathway strategy additionally targeting the TNF receptor super family 25 (TNFRSF25) using a fusion protein (TL1A-Ig) induces marked expansion of CD4+FoxP3+ Tregs systemically and within the ocular compartment. Importantly, these Tregs exhibit highly potent effector / suppressive activity. Preliminary data demonstrates that epigenetic regulation using a bromodomain inhibitor (BETi) can diminish pro-inflammatory cytokine production in vivo while simultaneously not interfering with Treg expansion and function. Excitingly in the context of this proposal, BETi significantly diminished clinical changes to the cornea when locally administered. We posit high-risk cornea is the ideal tissue to test this hypothesis because there is opportunity to locally modulate immunity and inflammation and we will apply elegant in vivo models to study, monitor and isolate antigen specific effector T cells and antigen specific Tregs using our newly developed B6-Nur77GFPFoxP3RFP mice. The studies proposed in this application will provide a unique opportunity to develop and test a 3-step combinatorial mechanism (CT-Cy, Treg, and BETi) for the establishment of allograft tolerance to maintain permanent HRVCT. Significantly, because HRVCT behave similarly to other vascularized organ transplants, work here can be applied to other tissues to induce transplant acceptance. Therefore, we will begin to test our approach through Treg manipulation in skin transplants first, using orthotopic mouse grafts and then continue by testing our strategy for translational application in humans by using human skin allografts in an immunodeficient mouse model and utilizing novel human reagents (mAb PTX-35) to manipulate human Treg cells. In total, this unique and powerful approach will bring together distinct immune regulatory mechanisms to induce tolerance to transplant antigens for the treatment of corneal blindness and other conditions requiring solid organ transplantation.